The invention relates to DNA sequences, recombinant DNA molecules and processes for producing annexin XI as well as methods for using annexin XI.
The annexins (or lipocortins) are a family of more than ten structurally related proteins. Annexins have been identified in a variety of eukaryotes from slime molds to mammals and higher plants, and the cDNAs encoding eight distinct mammalian annexins have been sequenced. All annexins have a conserved core domain and a highly variable amino terminal domain. The core domain consists of a 60-70 amino acid motif which is repeated four times in the smaller annexins (30-50 kD) and eight times in the larger annexins (.about.70 kD). The amino acid sequence of this core domain is 45 to 65% conserved between various annexins. For a given annexin the entire amino acid sequence is roughly 90% conserved between different mammalian species.
Annexins bind phospholipids and Ca.sup.2+ and associate with membrane preparations in a Ca.sup.2+ -dependent manner. Analysis of proteolytic fragments of several annexins indicates that the phospholipid and Ca.sup.2+ binding sites lie within the conserved core domain. Many, if not all, annexins inhibit phospholipase A.sub.2 (PLA.sub.2), an enzyme which releases fatty acids (e.g., arachidonic acid) esterified at the S.sub.N 2 position of membrane phospholipids. It is thought that annexins inhibit PLA.sub.2 activity by binding to phospholipids and blocking their interaction with PLA.sub.2. Arachidonic acid is a precursor in the synthesis of compounds, such as prostaglandins and leukotrienes, that are involved in inflammation. The lysophospholipids and their derivatives, such as platelet activating factor (PAF), may also promote inflammatory responses. Accordingly, it has been suggested that annexins may be useful for the treatment of inflammation. Purified annexins have been shown to have anti-inflammatory activity in vivo (Parente et al., Eur. J. Pharmacol. 99:233, 1984). Unlike many non-steroidal anti-inflammatory compounds, annexins have the potential to block an earlier step in the eicosanoid-generated production of inflammatory substances, via the cyclooxygenase pathway or the lipoxygenase pathway as well as via lysophospholipids and related mediators.
It has been suggested that annexins may have anti-coagulant activity. Factor X.sub.a -catalyzed activation of prothrombin, an important step in coagulation, is accelerated by protein cofactor V.sub.a and negatively charged phospholipids. Thus, annexins, by virtue of their ability to bind phospholipids, might inhibit prothrombin activation. Reutelingsperger et al. (Eur. J. Biochem. 173:171, 1988) describe an inhibitor of blood coagulation, VAC (annexin V), isolated from the intima of bovine aorta. This protein binds phospholipids in a Ca.sup.2+ -dependent manner. In a purified coagulation system this protein inhibited activation of prothrombin by factor X.sub.a.
Odenwald et al. (Biochem. and Biophys. Res. Comm. 112:147, 1983) disclose a 56 kD annexin-like protein (synexin II) isolated from bovine adrenal medulla and liver. Odenwald et al. report that synexin-II enhances Ca.sup.2+ -induced aggregation of chromaffin granule membranes and that this activity is protease resistant.
Towle et al. (Transactions of the 36th Annual Meeting of the Orthopaedic Research Society, February, 1990) report the use of antibodies raised against an anti-collagenase activator protein (stromelysin) to identify a clone in an interleukin-1 stimulated bovine chondrocyte CDNA expression library. Towle et al. report that partial sequence analysis of this clone predicts that it encodes a protein similar to lipocortins (annexins) I and II as well as PP4X (annexin IV). Northern analysis indicated that the steady state level of RNA capable of hybridizing to this cDNA clone increases four-fold upon exposure of chondrocytes to interleukin-1.
Towle et al. (Orthopaedic Trans. 14:343, 1990) describe a clone cp4x, isolated from a bovine cartilage cDNA library. A cartilage protein, immunologically cross-reactive with the fusion protein encoded by cp4x, co-purified (during initial purification steps) with other annexins. The clone encodes a protein that co-purifies with annexins and is homologous to pp4x (annexin IV). Towle et al. report that the steady state level of mRNA encoding cp4x is lower in interleukin-1 stimulated bovine chondrocytes than in non-stimulated chondrocytes.
Wallner et al. (U.S. Pat. No. 4,950,646) report the isolation of a cDNA encoding a 363 amino acid human lipocortin. E. coli or yeast transformed with a plasmid capable of expressing the lipocortin cDNA produce a 37 kD protein that is not produced in untransformed cells. Wallner et al. also report the isolation of a partial cDNA clone of a second lipocortin, N-lipocortin, which is homologous to their originally isolated lipocortin.
Wallner et al. (European Patent Application 0 330 396) report the isolation of cDNAs encoding human lipocortins III and V and the purification of bovine lipocortins IV and VI.
Huang et al. (J. Biol. Chem. 262:7639, 1989) report the identification of several proteolytic fragments of human lipocortin (annexin) I that are capable of inhibiting phospholipase A.sub.2. Huang et al. suggest that a region of lipocortin I from amino acids 83-346 may be important for lipocortin activity.